| With the widespread appearance of antibiotic-resistant bacteria, there is anincreasing demand for novel strategies to control infectious diseases. Every year morethan 80,000 Chinese die from antibiotics abuse, making China one of the worstoffenders in the word. It is presumed that we will be the first to enter the'post-antibiotic era' in comparison with other countries. Therefore, how to solve theproblem of bacterial resistance and development of new antibiotics have beenimminent.Pseudomonas aeruginosa (PA) is an opportunistic pathogen and it is a commoncause of serious hospital acquired infection. Infections can occur in any part of thebody, such as the wounds of bum, cornea, urinary tract and the lungs, etc. It is saidthat biofilm formation is the mean reason of antibiotic resistance. Biofilm is amulticellular, matrix-enclosed assemblies formed by bacteria and extracellular matrix(ECM). Once the biofilm formed, the antibiotic resistance will be increased more than100-1000 folds. Therefore, the interference of biofilm formation is of greatsignificance for the research and development of new antimicrobial drugs.Bacterial biofilm formation is controlled by complex net work. In the previousstudy, quorum sensing and the second messenger Cyclic-di-GMP have been reportedto relevant for biofilm formation. But the relationship of quorum sensing andC-di-GMP is unclear and this is the aim of our study. In this thesis, quorum sensingdeficient strain and C-di-GMP overexpression or degradation strain were used tostudy the relationship between both.The moving motility, virulence production and biofilm formation ability of quorumsensing mutant strains JP1 (â–³lasI) , pDO100 (â–³rhlI) , and MW (â–³lasIâ–³rhlI)were compared. The result indicated that the effect of Rhl-QS is more importantcompared with Las-QS. Rhl-QS dificient strain has lost the moving motility,rhamnolipid and pyocyanin production ability. Static and flow-cell models were used to investigate the effect of quorum sensing on biofilm formation. Contrast to flow-cellmodel, quorum sensing muatnt strain showed increased biofilm formation ability instatic condition. Biofilm formtion had increased the antibiotic resistance for about 100folds, but their was no obvious difference between the wild type and the quorumsensing mutant strain.A constitutive expression vector pMF-54A was constructed for constitutive expressof Diguanylate Cyclases (DGC) and Phosphodiesterases (PDE) and its stability was testedwith green fluorescent protein gene. After a full analysis of the whole genome ofPseudomonas aeruginosa PAO1, six proteins with GGDEF domain and four proteinswith EAL domain were chosen for next study. Target genes were recombinant intopMF-54A and transformed into PAO1. Static biofilm evaluation indicated thatphosphodiesterase PA2133 and diguanylate cyclase PA3702 significantly influence thebiofilm formation ability.Real time PCR analysis indicated that the transcription level increased 270 folds forPA2133 and 50 folds for PA3702. The virulence production, moving motility andbiofilm formation of wild type and quorum sensing muant strain with thoseoverexpress of PDE or DGC were observed. The results showed that the increase ofc-di-GMP inhibibited the moving motility of PAO1 and JP1 but improve theproduction of rhamnolipid. While the decrease of c-di-GMP can't restore the movingmotility and rhamnolipid production of both pDO100 and MW. In the static model,c-di-GMP increase the biofilm formation independent of quorum sensing.Furthermore, the increase of c-di-GMP level had improved the toxity of the strain.C-di-GMP can't change the antibiotic sensitivity of planktonic bacteria but theminimum biofilm eleberate concentration (MBEC) decreased for more than eightfolds after the transformation of PDE. Exogenous c-di-GMP inhibit the biofilmformation of PAO1. Real time PCR result indicated that c-di-GMP improved bothRhlI and RhlR transcription but there was no effect for LasI and LasR.The mechanisms of quorum sensing and c-di-GMP on biofilm formation wereinvestigated. The results indicated that quorum sensing deficiency strain, especiallyfor Rhl-QS deficient, inhibited the initial attachment but the microclone formation ability increased with long term incubation. The decrease of c-di-GMP cansignificantly inhibit the attachment ability, while the increase of c-di-GMP can'timprove the attachment. Strains with overexpress of DGC changed from vegetativegrowth phase to cryptic growth phase and with the elongation of the incubation timethey formed more microclones compared to wild type. We also evaluated the effect ofc-di-GMP on the exopolysaccharide production, hydophobicity and the cell surfacemotion organs. The result indicated that the hydrophobicity decreased, while thetranscription level of polysaccharide production gene pelA and pelD increased, withthe increase of c-di-GMP. Atomic Force Microscope and Transmission ElectronMicroscope observation doesn't show any differences in the motion organs.All in all, the two signal pathway was not completely isolated. The secondmessenger c-di-GMP can regulate Rhl-QS quorum sensing but its effect on biofilmformation was only partially depended on quorum sensing.
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